A fundamental gap exists in understanding how transcription by a translocating RNA polymerase modulates DNA topology and how transcription-coupled DNA supercoiling (TCDS) activates gene expression. For instance, the roles of sequence-specific DNA-binding proteins in TCDS are still not fully understood. The long- term goal of the proposed research is to understand how transcription affects DNA topology, chromosome structure, and the coupled DNA transactions, such as DNA replication and gene expression. The objectives of this application are to determine how certain sequence-specific DNA-binding proteins, such as bacteriophage lambda DNA replication initiator O protein and lactose repressor, regulate TCDS in vitro and in E. coli and to determine the mechanism by which TCDS activates gene expression. The central hypothesis is that the twin- supercoiled-domain model is the mechanism responsible for TCDS in which nucleoprotein complexes, especially those containing stable toroidal supercoils assembled from tightly-wrapping DNA around certain sequence-specific DNA-binding proteins, can form topological barriers that impede the diffusion and merger of independent chromosomal supercoil domains. In this case, the confined localized DNA supercoils may activate or inhibit the coupled DNA transactions. This hypothesis has been formulated on the basis of strong preliminary data produced in our laboratory and will be tested by pursuing four specific aims: 1) to determine the mechanisms by which certain sequence-specific DNA-binding proteins potently stimulate TCDS in the defined protein systems; 2) to study effects of the sequence-specific DNA-binding proteins on TCDS in E. coli; 3) to develop a novel system, based on a linear coliphage N15, to study activation of the Salmonella typhimurium leu-500 promoter by TCDS; 4) to establish a nationally competitive research program at Florida International University (the PI's development objective). This application will provide important knowledge for understanding the mechanism of TCDS and its roles in gene expression. It will also provide the necessary resources for the PI to transit to non-SCORE support within a four-year funding period. Public Health Relevance: The significance of this research stems from its potential to provide a basis for better understanding of an essential biological process: gene transcription and expression. It also provides a foundation for further understanding DNA topology, which plays an important role in genome stability and certain human hereditary diseases, such as fragile X syndrome and Huntington's disease.